4,4-Disubstituted piperidines, and methods of use thereof

ABSTRACT

One aspect of the present invention relates to heterocyclic compounds. A second aspect of the present invention relates to the use of the heterocyclic compounds as ligands for various mammalian cellular receptors, including dopamine transporters. The compounds of the present invention will find use in the treatment of numerous ailments, conditions and diseases which afflict mammals, including but not limited to addiction, anxiety, depression, sexual dysfunction, hypertension, migraine, Alzheimer&#39;s disease, obesity, emesis, psychosis, analgesia, schizophrenia, Parkinson&#39;s disease, restless leg syndrome, sleeping disorders, attention deficit hyperactivity disorder, irritable bowel syndrome, premature ejaculation, menstrual dysphoria syndrome, urinary incontinence, inflammatory pain, neuropathic pain, Lesche-Nyhane disease, Wilson&#39;s disease, and Tourette&#39;s syndrome. An additional aspect of the present invention relates to the synthesis of combinatorial libraries of the heterocyclic compounds, and the screening of those libraries for biological activity, e.g., in assays based on dopamine transporters.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/251,651, filed Dec. 6, 2000.

BACKGROUND OF THE INVENTION

Dopamine, norepinephrine and serotonin are mammalian monoamineneurotransmitters that play important roles in a wide variety ofphysiological processes. Therefore, compounds that selectively modulatethe activity of these three neurotransmitters, either individually, inpairs, or as a group, promise to serve as agents effective in thetreatment of a wide range of maladies, conditions and diseases thatafflict mammals due to atypical activities of these neurotransmitters.

For example, depression is believed to result from dysfunction in thenoradrenergic or serotonergic systems. Furthermore, the noradrenergicsystem appears to be associated with increased drive, whereas theserotonergic system relates more to changes in mood. Therefore, it ispossible that the different symptoms of depression may benefit fromdrugs acting mainly on one or the other of these neurotransmittersystems. On the other hand, a single compound that selectively affectsboth the noradrenergic and serotonergic systems should prove effectivein the treatment of depression comprising symptoms related todysfunction in both systems.

Dopamine plays a major role in addiction. Many of the concepts thatapply to dopamine apply to other neurotransmitters as well. As achemical messenger, dopamine is similar to adrenaline. Dopamine affectsbrain processes that control movement, emotional response, and abilityto experience pleasure and pain. Regulation of dopamine plays a crucialrole in our mental and physical health. Neurons containing theneurotransmitter dopamine are clustered in the midbrain in an areacalled the substantia nigra. In Parkinson's disease, thedopamine-transmitting neurons in this area die. As a result, the brainsof people with Parkinson's disease contain almost no dopamine. To helprelieve their symptoms, these patients are given L-DOPA, a drug that canbe converted in the brain to dopamine.

Certain drugs are known as dopamine agonists. These drugs bind todopamine receptors in place of dopamine and directly stimulate thosereceptors. Some dopamine agonists are currently used to treatParkinson's disease. These drugs can stimulate dopamine receptors evenin someone without dopamine-secreting neurons. In contrast to dopamineagonists, dopamine antagonists are drugs that bind but don't stimulatedopamine receptors. Antagonists can prevent or reverse the actions ofdopamine by keeping dopamine from activating receptors.

Dopamine antagonists are traditionally used to treat schizophrenia andrelated mental disorders. A person with schizophrenia may have anoveractive dopamine system. Dopamine antagonists can help regulate thissystem by “turning down” dopamine activity.

Cocaine and other drugs of abuse can alter dopamine function. Such drugsmay have very different actions. The specific action depends on whichdopamine receptors and brain regions the drugs stimulate or block, andhow well the compounds mimic dopamine. Drugs such as cocaine andamphetamine produce their effects by changing the flow ofneurotransmitters. These drugs are defined as indirect acting becausethey depend on the activity of neurons. In contrast, some drugs bypassneurotransmitters altogether and act directly on receptors. Such drugsare direct acting.

Use of these two types of drugs can lead to very different results intreating the same disease. As mentioned earlier, people with Parkinson'sdisease lose neurons that contain dopamine. To compensate for this loss,the body produces more dopamine receptors on other neurons. Indirectagonists are not very effective in treating the disease since theydepend on the presence of dopamine neurons. In contrast, direct agonistsare more effective because they stimulate dopamine receptors even whendopamine neurons are missing.

Certain drugs increase dopamine concentrations by preventing dopaminereuptake, leaving more dopamine in the synapse. An example is the widelyabused stimulant drug, cocaine. Another example is methylphenidate, usedtherapeutically to treat childhood hyperkinesis and symptoms ofnarcolepsy.

Sensitization or desensitization normally occur with drug exposure.However, addiction or mental illness can tamper with the reuptakesystem. This disrupts the normal levels of neurotransmitters in thebrain and can lead to faulty desensitization or sensitization. If thishappens in a region of the brain that serves emotion or motivation, theindividual can suffer severe consequences. For example, cocaine preventsdopamine reuptake by binding to proteins that normally transportdopamine. Not only does cocaine “bully” dopamine out of the way, it alsohangs on to the transport proteins much longer than dopamine does. As aresult, more dopamine remains to stimulate neurons, which causes aprolonged feelings of pleasure and excitement. Amphetamine alsoincreases dopamine levels. Again, the result is over-stimulation ofthese pleasure-pathway nerves in the brain.

Dopamine activity is implicated in the reinforcing effects of cocaine,amphetamine and natural rewards. However, dopamine abnormalities arealso believed to underlie some of the core attention deficits seen inacute schizophrenics.

Norepinephrine, also called noradrenaline, is a neurotransmitter thatdoubles part-time as a hormone. As a neurotransmitter, norepinephrinehelps to regulate arousal, dreaming, and moods. As a hormone, it acts toincrease blood pressure, constrict blood vessels and increase heartrate—responses that occur when we feel stress.

Serotonin (5-hydroxytryptamine, 5-HT) is widely distributed in animalsand plants, occurring in vertebrates, fruits, nuts, and venoms. A numberof congeners of serotonin are also found in nature and have been shownto possess a variety of peripheral and central nervous systemactivities. Serotonin may be obtained from a variety of dietary sources;however, endogenous 5-HT is synthesized in situ from tryptophan throughthe actions of the enzymes tryptophan hydroxylase and aromatic L-aminoacid decarboxylase. Both dietary and endogenous 5-HT are rapidlymetabolized and inactivated by monoamine oxidase and aldehydedehydrogenase to the major metabolite, 5-hydroxyindoleacetic acid(5-HIAA).

Serotonin is implicated in the etiology or treatment of variousdisorders, particularly those of the central nervous system, includinganxiety, depression, obsessive-compulsive disorder, schizophrenia,stroke, obesity, pain, hypertension, vascular disorders, migraine, andnausea. Recently, understanding of the role of 5-HT in these and otherdisorders has advanced rapidly due to increasing understanding of thephysiological role of various serotonin receptor subtypes.

It is currently estimated that up to 30% of clinically diagnosed casesof depression are resistant to all forms of drug therapy. To achieve aneffective therapy for such patients, it is logical to develop drugs thatpossess reuptake inhibition profiles different from those of drugscurrently available on the market. For example, the exact role ofdopamine in depressive illness is far from clear; however, interventionin the dopamine system may hold promise for the treatment of a subset ofmajor depression.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to heterocyclic compounds. Asecond aspect of the present invention relates to the use of theheterocyclic compounds as ligands for various mammalian cellularreceptors, including dopamine, serotonin, or norepinephrinetransporters. The compounds of the present invention will find use inthe treatment of numerous ailments, conditions and diseases whichafflict mammals, including but not limited to addiction, anxiety,depression, sexual dysfunction, hypertension, migraine, Alzheimer'sdisease, obesity, emesis, psychosis, analgesia, schizophrenia,Parkinson's disease, restless leg syndrome, sleeping disorders,attention deficit hyperactivity disorder, irritable bowel syndrome,premature ejaculation, menstrual dysphoria syndrome, urinaryincontinence, inflammatory pain, neuropathic pain, Lesche-Nyhanedisease, Wilson's disease, and Tourette's syndrome.

An additional aspect of the present invention relates to the synthesisof combinatorial libraries of the heterocyclic compounds, and thescreening of those libraries for biological activity, e.g., in assaysbased on dopamine, serotonin or norepinephrine transporters.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a synthetic scheme used to synthesize a compound of thepresent invention.

FIG. 2 depicts a compound of the present invention, and its activity incertain assays based on neurotransmitter transporters.

FIG. 3 depicts various compounds of the present invention and their IC₅₀values against mammalian norepinephrine, dopamine, and5-hydroxytryptophan transporters.

FIG. 4 depicts various compounds of the present invention and their IC₅₀values against mammalian norepinephrine, dopamine, and5-hydroxytryptophan transporters.

FIG. 5 depicts various compounds of the present invention and their IC₅₀values against mammalian norepinephrine, dopamine, and5-hydroxytryptophan transporters.

FIG. 6 depicts various compounds of the present invention and their IC₅₀values against mammalian norepinephrine, dopamine, and5-hydroxytryptophan transporters.

FIG. 7 depicts various compounds of the present invention and their EC₅₀values against a mammalian dopamine transporter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides heterocyclic compounds. Furthermore, thepresent invention provides heterocyclic compounds that are ligands fordopamine, serotonin, or norepinephrine receptors or transporters, andmethods of use thereof for the treatment of numerous ailments,conditions and diseases which afflict mammals, including but not limitedto addiction, anxiety, depression, sexual dysfunction, hypertension,migraine, Alzheimer's disease, obesity, emesis, psychosis, analgesia,schizophrenia, Parkinson's disease, restless leg syndrome, sleepingdisorders, attention deficit hyperactivity disorder, irritable bowelsyndrome, premature ejaculation, menstrual dysphoria syndrome, urinaryincontinence, inflammatory pain, neuropathic pain, Lesche-Nyhanedisease, Wilson's disease, and Tourette's syndrome. An additional aspectof the present invention relates to the synthesis of combinatoriallibraries of the heterocyclic compounds, and the screening of thoselibraries for biological activity, e.g., in assays based on dopaminetransporters. The present invention also relates to pharmaceuticalformulations of the heterocyclic compounds.

In certain embodiments, compounds of the present invention are ligandsfor mammalian receptors for dopamine, norepinephrine, serotonin, any twoof these three neurotransmitters or all of them. In certain embodiments,compounds of the present invention are ligands for mammaliantransporters of dopamine, norepinephrine, serotonin, any two of thesethree neurotransmitters or all of them. In certain embodiments,compounds of the present invention are agonists of mammalian receptorsfor dopamine, norepinephrine, serotonin, any two of these threeneurotransmitters or all of them. In certain embodiments, compounds ofthe present invention are antagonists or inverse agonists of mammalianreceptors for dopamine, norepinephrine, serotonin, any two of thesethree neurotransmitters or all of them. In certain embodiments,compounds of the present invention are agonists of mammaliantransporters of dopamine, norepinephrine, serotonin, any two of thesethree neurotransmitters or all of them. In certain embodiments,compounds of the present invention are antagonists or inverse agonistsof mammalian transporters of dopamine, norepinephrine, serotonin, anytwo of these three neurotransmitters or all of them.

In certain embodiments, compounds of the present invention are ligandsfor mammalian dopamine receptors. In certain embodiments, compounds ofthe present invention are ligands for mammalian dopamine transporters.In certain embodiments, compounds of the present invention are agonistsof mammalian dopamine receptors. In certain embodiments, compounds ofthe present invention are antagonists or inverse agonists of mammaliandopamine receptors. In certain embodiments, compounds of the presentinvention are agonists of mammalian dopamine transporters. In certainembodiments, compounds of the present invention are antagonists orinverse agonists of mammalian dopamine transporters.

The mammalian dopamine receptor or transporter is a member of a familyof cell surface proteins that permit intracellular transduction ofextracellular signals. Cell surface proteins provide eukaryotic andprokaryotic cells a means to detect extracellular signals and transducesuch signals intracellularly in a manner that ultimately results in acellular response or a concerted tissue or organ response. Cell surfaceproteins, by intracellularly transmitting information regarding theextracellular environment via specific intracellular pathways induce anappropriate response to a particular stimulus. The response may beimmediate and transient, slow and sustained, or some mixture thereof. Byvirtue of an array of varied membrane surface proteins, eukaryotic cellsare exquisitely sensitive to their environment.

Extracellular signal molecules, such as growth hormones, vasodilatorsand neurotransmitters, exert their effects, at least in part, viainteraction with cell surface proteins. For example, some extracellularsignal molecules cause changes in transcription of target gene viachanges in the levels of secondary messengers, such as cAMP. Othersignals, indirectly alter gene expression by activating the expressionof genes, such as immediate-early genes that encode regulatory proteins,which in turn activate expression of other genes that encodetranscriptional regulatory proteins. For example, neuron gene expressionis modulated by numerous extracellular signals, includingneurotransmitters and membrane electrical activity. Transsynapticsignals cause rapid responses in neurons that occur over a period oftime ranging from milliseconds, such as the opening of ligand-gatedchannels, to seconds and minutes, such as second messenger-mediatedevents. Genes in neural cells that are responsive to transsynapticstimulation and membrane electrical activity, include genes, calledimmediate early genes, whose transcription is activated rapidly, withinminutes, and transiently (see, e.g., Sheng et al. (1990) Neuron 4:477-485), and genes whose expression requires protein synthesis andwhose expression is induced or altered over the course of hours.

Cell surface receptors and ion channels are among the cell surfaceproteins that respond to extracellular signals and initiate the eventsthat lead to this varied gene expression and response. Ion channels andcell surface-localized receptors are ubiquitous and physiologicallyimportant cell surface membrane proteins. They play a central role inregulating intracellular levels of various ions and chemicals, many ofwhich are important for cell viability and function.

Cell surface-localized receptors are membrane spanning proteins thatbind extracellular signalling molecules or changes in the extracellularenvironment and transmit the signal via signal transduction pathways toeffect a cellular response. Cell surface receptors bind circulatingsignal polypeptides, such as neurotransmitters, growth factors andhormones, as the initiating step in the induction of numerousintracellular pathways. Receptors are classified on the basis of theparticular type of pathway that is induced. Included among these classesof receptors are those that bind growth factors and have intrinsictyrosine kinase activity, such as the heparin binding growth factor(HBGF) receptors, and those that couple to effector proteins throughguanine nucleotide binding regulatory proteins, which are referred to asG protein coupled receptors and G proteins, respectively.

The G protein transmembrane signaling pathways consist of threeproteins: receptors, G proteins and effectors. G proteins, which are theintermediaries in transmembrane signaling pathways, are heterodimers andconsist of alpha, beta and gamma subunits. Among the members of a familyof G proteins the alpha subunits differ. Functions of G proteins areregulated by the cyclic association of GTP with the alpha subunitfollowed by hydrolysis of GTP to GDP and dissociation of GDP.

G protein coupled receptors are a diverse class of receptors thatmediate signal transduction by binding to G proteins. Signaltransduction is initiated via ligand binding to the cell membranereceptor, which stimulates binding of the receptor to the G protein. Thereceptor G protein interaction releases GDP, which is specifically boundto the G protein, and permits the binding of GTP, which activates the Gprotein. Activated G protein dissociates from the receptor and activatesthe effector protein, which regulates the intracellular levels ofspecific second messengers. Examples of such effector proteins includeadenyl cyclase, guanyl cyclase, phospholipase C, and others.

G protein-coupled receptors, which are glycoproteins, are known to sharecertain structural similarities and homologies (see, e-g., Gilman, A.G., Ann. Rev. Biochem.56: 615-649 (1987), Strader, C. D. et al. TheFASEB Journal 3: 1825-1832 (1989), Kobilka, B. K., et al. Nature329:75-79 (1985) and Young et al. Cell 45: 711-719 (1986)). Among the Gprotein-coupled receptors that have been identified and cloned are thesubstance P receptor, the angiotensin receptor, the alpha- andbeta-adrenergic receptors and the serotonin receptors. G protein-coupledreceptors share a conserved structural motif. The general and commonstructural features of the G protein-coupled receptors are the existenceof seven hydrophobic stretches of about 20-25 amino acids eachsurrounded by eight hydrophilic regions of variable length. It has beenpostulated that each of the seven hydrophobic regions forms atransmembrane alpha helix and the intervening hydrophilic regions formalternately intracellularly and extracellularly exposed loops. The thirdcytosolic loop between transmembrane domains five and six is theintracellular domain responsible for the interaction with G proteins.

G protein-coupled receptors are known to be inducible. This inducibilitywas originally described in lower eukaryotes. For example, the cAMPreceptor of the cellular slime mold, Dictyostelium, is induced duringdifferentiation (Klein et al., Science 241: 1467-1472 (1988). During theDictyostelium discoideum differentiation pathway, cAMP, induces highlevel expression of its G protein-coupled receptor. This receptortransduces the signal to induce the expression of the other genesinvolved in chemotaxis, which permits multicellular aggregates to align,organize and form stalks (see, Firtel, R. A., et al. Cell 58: 235-239(1989) and Devreotes, P., Science 245: 1054-1058 (1989)).

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “cell surface proteins” includes molecules that occur on thesurface of cells, interact with the extracellular environment, andtransmit or transduce information regarding the environmentintracellularly.

The term “extracellular signals” includes a molecule or a change in theenvironment that is transduced intracellularly via cell surface proteinsthat interact, directly or indirectly, with the signal. An extracellularsignal is any compound or substance that in some manner specificallyalters the activity of a cell surface protein. Examples of such signalsinclude, but are not limited to, molecules such as acetylcholine, growthfactors, hormones and other mitogenic substances, such as phorbolmistric acetate (PMA), that bind to cell surface receptors and ionchannels and modulate the activity of such receptors and channels.Extracellular signals also includes as yet unidentified substances thatmodulate the activity of a cell surface protein and thereby affectintracellular functions and that are potential pharmacological agentsthat may be used to treat specific diseases by modulating the activityof specific cell surface receptors.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect. Alternatively, the dose which produces apre-determined response in 50% of test subjects or preparations.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The term “structure-activity relationship (SAR)” refers to the way inwhich altering the molecular structure of drugs alters their interactionwith a receptor, enzyme, etc.

The term “agonist” refers to a compound that mimics the action ofnatural transmitter or, when the natural transmitter is not known,causes changes at the receptor complex in the absence of other receptorligands.

The term “antagonist” refers to a compound that binds to a receptorsite, but does not cause any physiological changes unless anotherreceptor ligand is present.

The term “inverse agonist” refers to a compound that binds to aconstitutively active receptor site and reduces its physiologicalfunction.

The term “competitive antagonist” refers to a compound that binds to areceptor site; its effects can be overcome by increased concentration ofthe agonist.

The term “partial agonist” refers to a compound that binds to a receptorsite but does not produce the maximal effect regardless of itsconcentration.

The term “ligand” refers to a compound that binds at the receptor site.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (a)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=−0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl, formyl, sulfonyl,trifluoromethyl, cyano, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′11 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula: in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as analgesics), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound in binding to sigmareceptors. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Compounds of the Invention

In certain embodiments, the present invention relates to a compoundrepresented by A:

wherein

R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl, heteroaryl,acyl, or sulfonyl;

R₁ represents aryl, or heteroaryl;

R₂ represents RO-alkyl, (R)₂N-alkyl, RS-alkyl, RO-cycloalkyl,(R)₂N-cycloalkyl, or RS-cycloalkyl;

R₃ represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, —OR, or F;

R₄ represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, —OR, or F;

R₅ represents an aryl or heteroaryl group;

R₃ and R₄ may be connected through a covalent bond;

n is 0, 1, or 2; and

the stereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein R₁ representsaryl.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein R₂ representsRO-alkyl.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; and R₃represents H, alkyl, or F.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; and R₄represents H, alkyl, or F.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 0; and R₅repesents phenyl or thiophene.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein R₅ represents asubstituted phenyl; and R₁ represents aryl.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein R₅ represents asubstituted phenyl; R₁ represents aryl; and R₂ represents RO-alkyl.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; R₅represents a substituted phenyl; R₁ represents aryl; R₂ representsRO-alkyl; and R₄ represents H, alkyl, or F.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; R₅represents a substituted phenyl; R₁ represents aryl; R₂ representsRO-alkyl; R₃ represents H, alkyl, or F; and R₄ represents H, alkyl, orF.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; and R₃and R₄ are joined through a covalent bond to form a cyclopropyl ring.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; and R₃and R₄ are joined through a covalent bond to form a cyclobutyl ring.

In certain embodiments, the compounds of the present invention arerepresented by A and the attendant definitions, wherein n is 1; and R₃and R₄ are joined through a covalent bond to form a cyclopentyl ring.

In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined above, wherein saidcompound is a single stereoisomer.

In assays based on mammalian dopamine, serotonin, or norepinephrinereceptors or transporters, certain compounds according to structure Ahave IC₅₀ values less than 1 μM, more preferably less than 100 nM, andmost preferably less than 10 nM.

In assays based on mammalian dopamine receptors or transporters, certaincompounds according to structure A have IC₅₀ values less than 1 μM, morepreferably less than 100 nM, and most preferably less than 10 nM.

In assays based on mammalian dopamine, serotonin, or norepinephrinereceptors or transporters, certain compounds according to structure Ahave EC₅₀ values less than 1 μM, more preferably less than 100 nM, andmost preferably less than 10 nM.

In assays based on mammalian dopamine receptors or transporters, certaincompounds according to structure A have EC₅₀ values less than 1 μM, morepreferably less than 100 nM, and most preferably less than 10 nM.

In certain embodiments, the present invention relates to a formulation,comprising a compound represented by any of the structures outlinedabove; and a pharmaceutically acceptable excipient.

In certain embodiments, the present invention relates to ligands forreceptors or transporters of dopamine, serotonin, or norepinephrine,wherein the ligands are represented by any of the structures outlinedabove, and any of the sets of definitions associated with one of thosestructures. In certain embodiments, the ligands of the present inventionare antagonists or agonists of receptors or transporters of dopamine,serotonin, or norepinephrine. In any event, the ligands of the presentinvention preferably exert their effect on the dopamine, serotonin, ornorepinephrine receptors or transporters at a concentration less thanabout 1 micromolar, more preferably at a concentration less than about100 nanomolar, and most preferably at a concentration less than 10nanomolar.

The present invention contemplates pharmaceutical formulations of theligands of the present invention. In certain embodiments, thepharmaceutical formulations will comprise ligands of the presentinvention that selectively effect dopamine receptors or transporters,and thereby have a therapeutic effect on an acute or chronic ailment,disease or malady that is at least in part due to biochemical orphysiological processes associated with dopamine receptors ortransporters. The Background of the Invention (see above) teachesexamples of acute or chronic ailments, diseases or maladies that arecaused or exacerbated by biochemical or physiological processesassociated with dopamine receptors or transporters. One of ordinaryskill in the art will be able to accumulate, by reference to thescientific literature, a more comprehensive list of acute or chronicailments, diseases or maladies that are caused or exacerbated bybiochemical or physiological processes associated with dopaminereceptors or transporters. The present invention contemplatespharmaceutical formulations of ligands of the present invention thatwill be of medicinal value against the aforementioned acute or chronicailments, diseases or maladies.

Methods of the Invention

In certain embodiments, the present invention relates to a method ofmodulating the activity of a dopamine, serotonin, or norepinephrinereceptor or transporter in a mammal, comprising the step ofadministering to said mammal a therapeutically effective amount of acompound represented by A:

wherein

R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl, heteroaryl,acyl, or sulfonyl;

R₁ represents aryl, heteroaryl, aralkyl, or heteroaralkyl;

R₂ represents alkyl, RO-alkyl, (R)₂N-alkyl, RS-alkyl, cycloalkyl,RO-cycloalkyl, (R)₂N-cycloalkyl, RS-cycloalkyl, alkenyl, aryl, orheteroaryl;

R₄ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

R₅ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

any geminal or vicinal pairs of R₄ and R₅ may be connected through acovalent bond;

n is independently for each occurrence 0, 1, 2, 3, or 4; and

the stereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to a method ofmodulating the activity of a dopamine receptor or transporter in amammal, comprising the step of administering to said mammal atherapeutically effective amount of a compound represented by A:

wherein

R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl, heteroaryl,acyl, or sulfonyl;

R₁ represents aryl, heteroaryl, aralkyl, or heteroaralkyl;

R₂ represents alkyl, RO-alkyl, (R)₂N-alkyl, RS-alkyl, cycloalkyl,RO-cycloalkyl, (R)₂N-cycloalkyl, RS-cycloalkyl, alkenyl, aryl, orheteroaryl;

R₄ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

R₅ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

any geminal or vicinal pairs of R₄ and R₅ may be connected through acovalent bond;

n is independently for each occurrence 0, 1, 2, 3, or 4; and

the stereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to a method oftreating a mammal suffering from addiction, anxiety, depression, sexualdysfunction, hypertension, migraine, Alzheimer's disease, obesity,emesis, psychosis, analgesia, schizophrenia, Parkinson's disease,restless leg syndrome, sleeping disorders, attention deficithyperactivity disorder, irritable bowel syndrome, premature ejaculation,menstrual dysphoria syndrome, urinary incontinence, inflammatory pain,neuropathic pain, Lesche-Nyhane disease, Wilson's disease, or Tourette'ssyndrome, comprising the step of administering to said mammal atherapeutically effective amount of a compound represented by A:

wherein

R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl, heteroaryl,acyl, or sulfonyl;

R₁ represents aryl, heteroaryl, aralkyl, or heteroaralkyl;

R₂ represents alkyl, RO-alkyl, (R)₂N-alkyl, RS-alkyl, cycloalkyl,RO-cycloalkyl, (R)₂N-cycloalkyl, RS-cycloalkyl, alkenyl, aryl, orheteroaryl;

R₄ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

R₅ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;

any geminal or vicinal pairs of R₄ and R₅ may be connected through acovalent bond;

n is independently for each occurrence 0, 1, 2, 3, or 4; and

the stereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations.

Biochemical Activity at Cellular Receptors, and Assays to Detect ThatActivity

Assaying processes are well known in the art in which a reagent is addedto a sample, and measurements of the sample and reagent are made toidentify sample attributes stimulated by the reagent. For example, onesuch assay process concerns determining in a chromogenic assay theamount of an enzyme present in a biological sample or solution. Suchassays are based on the development of a colored product in the reactionsolution. The reaction develops as the enzyme catalyzes the conversionof a colorless chromogenic substrate to a colored product.

Another assay useful in the present invention concerns determining theability of a ligand to bind to a biological receptor utilizing atechnique well known in the art referred to as a radioligand bindingassay. This assay accurately determines the specific binding of aradioligand to a targeted receptor through the delineation of its totaland nonspecific binding components. Total binding is defined as theamount of radioligand that remains following the rapid separation of theradioligand bound in a receptor preparation (cell homogenates orrecombinate receptors) from that which is unbound. The nonspecificbinding component is defined as the amount of radioligand that remainsfollowing separation of the reaction mixture consisting of receptor,radioligand and an excess of unlabeled ligand. Under this condition, theonly radioligand that remains represents that which is bound tocomponents other that receptor. The specific radioligand bound isdetermined by subtracting the nonspecific from total radioactivitybound. For a specific example of radioligand binding assay for μ-opioidreceptor, see Wang, J. B. et al. FEBS Letters 1994, 338, 217.

Assays useful in the present invention concern determining the activityof receptors the activation of which initiates subsequent intracellularevents in which intracellular stores of calcium ions are released foruse as a second messenger. Activation of some G-protein-coupledreceptors stimulates the formation of inositol triphosphate (IP3, aG-protein-coupled receptor second messenger) through phospholipaseC-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine(1984). Nature 312:315-21. IP3 in turn stimulates the release ofintracellular calcium ion stores.

A change in cytoplasmic calcium ion levels caused by release of calciumions from intracellular stores is used to determine G-protein-coupledreceptor function. This is another type of indirect assay. AmongG-protein-coupled receptors are muscarinic acetylcholine receptors(mAChR), adrenergic receptors, sigma receptors, serotonin receptors,dopamine receptors, angiotensin receptors, adenosine receptors,bradykinin receptors, metabotropic excitatory amino acid receptors andthe like. Cells expressing such G-protein-coupled receptors may exhibitincreased cytoplasmic calcium levels as a result of contribution fromboth intracellular stores and via activation of ion channels, in whichcase it may be desirable although not necessary to conduct such assaysin calcium-free buffer, optionally supplemented with a chelating agentsuch as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores. Another type of indirect assayinvolves determining the activity of receptors which, when activated,result in a change in the level of intracellular cyclic nucleotides,e.g., cAMP, cGMP. For example, activation of some dopamine, serotonin,metabotropic glutamate receptors and muscarinic acetylcholine receptorsresults in a decrease in the cAMP or cGMP levels of the cytoplasm.

Furthermore, there are cyclic nucleotide-gated ion channels, e.g., rodphotoreceptor cell channels and olfactory neuron channels [see,Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872and Dhallan et al. (1990) Nature 347:184-187] that are permeable tocations upon activation by binding of cAMP or cGMP. A change incytoplasmic ion levels caused by a change in the amount of cyclicnucleotide activation of photo-receptor or olfactory neuron channels isused to determine function of receptors that cause a change in cAMP orcGMP levels when activated. In cases where activation of the receptorresults in a decrease in cyclic nucleotide levels, it may be preferableto expose the cells to agents that increase intracellular cyclicnucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay. Cell for thistype of assay can be made by co-transfection of a host cell with DNAencoding a cyclic nucleotide-gated ion channel and a DNA encoding areceptor (e.g., certain metabotropic glutamate receptors, muscarinicacetylcholine receptors, dopamine receptors, serotonin receptors and thelike, which, when activated, causes a change in cyclic nucleotide levelsin the cytoplasm.

Any cell expressing a receptor protein which is capable, uponactivation, of directly increasing the intracellular concentration ofcalcium, such as by opening gated calcium channels, or indirectlyaffecting the concentration of intracellular calcium as by causinginitiation of a reaction which utilizes Ca<2+> as a second messenger(e.g., G-protein-coupled receptors), may form the basis of an assay.Cells endogenously expressing such receptors or ion channels and cellswhich may be transfected with a suitable vector encoding one or moresuch cell surface proteins are known to those of skill in the art or maybe identified by those of skill in the art. Although essentially anycell which expresses endogenous ion channel and/or receptor activity maybe used, it is preferred to use cells transformed or transfected withheterologous DNAs encoding such ion channels and/or receptors so as toexpress predominantly a single type of ion channel or receptor. Manycells that may be genetically engineered to express a heterologous cellsurface protein are known. Such cells include, but are not limited to,baby hamster kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCCNo. CCLI.3), DG44 cells [see, Chasin (1986) Cell. Molec. Genet. 12:555]human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamsterovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCCNo. CRL1721) and COS-7 cells (ATCC No. CRL1651). Preferred cells forheterologous cell surface protein expression are those that can bereadily and efficiently transfected. Preferred cells include HEK 293cells, such as those described in U.S. Pat. No. 5,024,939.

Any compound which is known to activate ion channels or receptors ofinterest may be used to initiate an assay. Choosing an appropriate ionchannel- or receptor-activating reagent depending on the ion channel orreceptor of interest is within the skill of the art. Directdepolarization of the cell membrane to determine calcium channelactivity may be accomplished by adding a potassium salt solution havinga concentration of potassium ions such that the final concentration ofpotassium ions in the cell-containing well is in the range of about50-150 mM (e.g., 50 mM KCl). With respect to ligand-gated receptors andligand-gated ion channels, ligands are known which have affinity for andactivate such receptors. For example, nicotinic acetyloholine receptorsare known to be activated by nicotine or acetylcholine; similarly,muscarinic acetylcholine receptors may be activated by addition ofmuscarine or carbamylcholine.

Agonist assays may be carried out on cells known to possess ion channelsand/or receptors to determine what effect, if any, a compound has onactivation or potentiation of ion channels or receptors of interest.Agonist assays also may be carried out using a reagent known to possession channel- or receptor-activating capacity to determine whether a cellexpresses the respective finctional ion channel or receptor of interest.

Contacting a functional receptor or ion channel with agonist typicallyactivates a transient reaction; and prolonged exposure to an agonist maydesensitize the receptor or ion channel to subsequent activation. Thus,in general, assays for determining ion channel or receptor functionshould be initiated by addition of agonist (i.e., in a reagent solutionused to initiate the reaction). The potency of a compound having agonistactivity is determined by the detected change in some observable in thecells (typically an increase, although activation of certain receptorscauses a decrease) as compared to the level of the observable in eitherthe same cell, or substantially identical cell, which is treatedsubstantially identically except that reagent lacking the agonist (i.e.,control) is added to the well. Where an agonist assay is performed totest whether or not a cell expresses the functional receptor or ionchannel of interest, known agonist is added to test-cell-containingwells and to wells containing control cells (substantially identicalcell that lacks the specific receptors or ion channels) and the levelsof observable are compared. Depending on the assay, cells lacking theion channel and/or receptor of interest should exhibit substantially noincrease in observable in response to the known agonist. A substantiallyidentical cell may be derived from the same cells from which recombinantcells are prepared but which have not been modified by introduction ofheterologous DNA. Alternatively, it may be a cell in which the specificreceptors or ion channels are removed. Any statistically or otherwisesignificant difference in the level of observable indicates that thetest compound has in some manner altered the activity of the specificreceptor or ion channel or that the test cell possesses the specificfinctional receptor or ion channel.

In an example of drug screening assays for identifying compounds whichhave the ability to modulate ion channels or receptors of interest,individual wells (or duplicate wells, etc.) contain a distinct celltype, or distinct recombinant cell line expressing a homogeneouspopulation of a receptor or ion channel of interest, so that thecompound having unidentified activity may be screened to determinewhether it possesses modulatory activity with respect to one or more ofa variety of functional ion channels or receptors. It is alsocontemplated that each of the individual wells may contain the same celltype so that multiple compounds (obtained from different reagent sourcesin the apparatus or contained within different wells) can be screenedand compared for modulating activity with respect to one particularreceptor or ion channel type.

Antagonist assays, including drug screening assays, may be carried outby incubating cells having functional ion channels and/or receptors inthe presence and absence of one or more compounds, added to the solutionbathing the cells in the respective wells of the microtiter plate for anamount of time sufficient (to the extent that the compound has affinityfor the ion channel and/or receptor of interest) for the compound(s) tobind to the receptors and/or ion channels, then activating the ionchannels or receptors by addition of known agonist, and measuring thelevel of observable in the cells as compared to the level of observablein either the same cell, or substantially identical cell, in the absenceof the putative antagonist.

The assays are thus useful for rapidly screening compounds to identifythose that modulate any receptor or ion channel in a cell. Inparticular, assays can be used to test functional ligand-receptor orligand-ion channel interactions for cell receptors includingligand-gated ion channels, voltage-gated ion channels, G-protein-coupledreceptors and growth factor receptors.

Those of ordinary skill in the art will recognize that assays mayencompass measuring a detectable change of a solution as a consequenceof a cellular event which allows a compound, capable of differentialcharacteristics, to change its characteristics in response to thecellular event. By selecting a particular compound which is capable ofdifferential characteristics upon the occurrence of a cellular event,various assays may be performed. For example, assays for determining thecapacity of a compound to induce cell injury or cell death may becarried out by loading the cells with a pH-sensitive fluorescentindicator such as BCECF (Molecular Probes, Inc., Eugene, Oreg. 97402,Catalog #B1150) and measuring cell injury or cell death as a function ofchanging fluorescence over time.

In a further example of useful assays, the function of receptors whoseactivation results in a change in the cyclic nucleotide levels of thecytoplasm may be directly determined in assays of cells that expresssuch receptors and that have been injected with a fluorescent compoundthat changes fluorescence upon binding cAMP. The fluorescent compoundcomprises cAMP-dependent-protein kinase in which the catalytic andregulatory subunits are each labelled with a different fluorescent-dye[Adams et al. (1991) Nature 349:694-697]. When cAMP binds to theregulatory subunits, the fluorescence emission spectrum changes; thischange can be used as an indication of a change in cAMP concentration.

The function of certain neurotransmitter transporters which are presentat the synaptic cleft at the junction between two neurons may bedetermined by the development of fluorescence in the cytoplasm of suchneurons when conjugates of an amine acid and fluorescent indicator(wherein the fluorescent indicator of the conjugate is an acetoxymethylester derivative e.g., 5-(aminoacetamido)fluorescein; Molecular Probes,Catalog #A1363) are transported by the neurotransmitter transporter intothe cytoplasm of the cell where the ester group is cleaved by esteraseactivity and the conjugate becomes fluorescent.

In practicing an assay of this type, a reporter gene construct isinserted into an eukaryotic cell to produce a recombinant cell which haspresent on its surface a cell surface protein of a specific type. Thecell surface receptor may be endogenously expressed or it may beexpressed from a heterologous gene that has been introduced into thecell. Methods for introducing heterologous DNA into eukaryotic cellsare-well known in the art and any such method may be used. In addition,DNA encoding various cell surface proteins is known to those of skill inthe art or it may be cloned by any method known to those of skill in theart.

The recombinant cell is contacted with a test compound and the level ofreporter gene expression is measured. The contacting may be effected inany vehicle and the testing may be by any means using any protocols,such as serial dilution, for assessing specific molecular interactionsknown to those of skill in the art. After contacting the recombinantcell for a sufficient time to effect any interactions, the level of geneexpression is measured. The amount of time to effect such interactionsmay be empirically determined, such as by running a time course andmeasuring the level of transcription as a function of time. The amountof transcription may be measured using any method known to those ofskill in the art to be suitable. For example, specific mRNA expressionmay be detected using Northern blots or specific protein product may beidentified by a characteristic stain. The amount of transcription isthen compared to the amount of transcription in either the same cell inthe absence of the test. compound or it may be compared with the amountof transcription in a substantially identical cell that lacks thespecific receptors. A substantially identical cell may be derived fromthe same cells from which the recombinant cell was prepared but whichhad not been modified by introduction of heterologous DNA.Alternatively, it may be a cell in which the specific receptors areremoved. Any statistically or otherwise significant difference in theamount of transcription indicates that the test compound has in somemanner altered the activity of the specific receptor.

If the test compound does not appear to enhance, activate or induce theactivity of the cell surface protein, the assay may be repeated andmodified by the introduction of a step in which the recombinant cell isfirst tested for the ability of a known agonist or activator of thespecific receptor to activate transcription if the transcription isinduced, the test compound is then assayed for its ability to inhibit,block or otherwise affect the activity of the agonist.

The transcription based assay is useful for identifying compounds thatinteract with any cell surface protein whose activity ultimately altersgene expression. In particular, the assays can be used to testfunctional ligand-receptor or ligand-ion channel interactions for anumber of categories of cell surface-localized receptors, including:ligand-gated ion channels and voltage-gated ion channels, and Gprotein-coupled receptors.

Any transfectable cell that can express the desired cell surface proteinin a manner such the protein functions to intracellularly transduce anextracellular signal may be used. The cells may be selected such thatthey endogenously express the cell surface protein or may be geneticallyengineered to do so. Many such cells are known to those of skill in theart. Such cells include, but are not limited to Ltk<−> cells, PC12 cellsand COS-7 cells.

The preparation of cells which express a receptor or ion channel and areporter gene expression construct, and which are useful for testingcompounds to assess their activities, is exemplified in the Examplesprovided herewith by reference to mammalian Ltk<−> and COS-7 cell lines,which express the Type I human muscarinic (HM1) receptor and which aretransformed with either a c-fos promoter-CAT reporter gene expressionconstruct or a c-fos promoter-luciferase reporter gene expressionconstruct.

Any cell surface protein that is known to those of skill in the art orthat may be identified by those of skill in the art may used in theassay. The cell surface protein may endogenously expressed on theselected cell or it may be expressed from cloned DNA. Exemplary cellsurface proteins include, but are not limited to, cell surface receptorsand ion channels. Cell surface receptors include, but are not limitedto, muscarinic receptors (e.g., human M2 (GenBank accession #M16404);rat M3 (GenBank accession #M16407); human M4 (GenBank accession#M16405); human M5 (Bonner et al. (1988) Neuron 1:403-410); and thelike); neuronal nicotinic acetylcholine receptors (e.g., the alpha 2,alpha 3 and beta 2 subtypes disclosed in U.S. Ser. No. 504,455 (filedApr. 3, 1990), hereby expressly incorporated by reference herein in itsentirety); the rat alpha 2 subunit (Wada et al. (1988) Science240:330-334); the rat alpha 3 subunit (Boulter et al. (1986) Nature319:368-374); the rat alpha 4 subunit (Goldman et al. (1987) cell48:965-973); the rat alpha 5 subunit (Boulter et al. (1990) J. Biol.Chem. 265:4472-4482); the rat beta 2 subunit (Deneris et al. (1988)Neuron 1:45-54); the rat beta 3 subunit (Deneris et al. (1989) J. Biol.Chem. 264: 6268-6272); the rat beta 4 subunit (Duvoisin et al. (1989)Neuron 3:487-496); combinations of the rat alpha subunits, beta subunitsand alpha and beta subunits; GABA receptors (e.g., the bovine alpha 1and beta 1 subunits (Schofield et al. (1987) Nature 328:221-227); thebovine alpha 2 and alpha 3 subunits (Levitan et al. (1988) Nature335:76-79); the gamma-subunit (Pritchett et al. (1989) Nature338:582-585); the beta 2 and beta 3 subunits (Ymer et alo (1989) EMBO J.8:1665-1670); the delta subunit (Shivers, B.D. (1989) Neuron 3:327-337);and the like); glutamate receptors (e.g., receptor isolated from ratbrain (Hollmann et al. (1989) Nature 342:643-648); and the like);adrenergic receptors (e.g., human beta 1 (Frielle et al. (1987) Proc.Natl. Acad. Sci. 84.:7920-7924); human alpha 2 (Kobilka et al. (1987)Science 238:650-656); hamster beta 2 (Dixon et al. (1986) Nature321:75-79); and the like); dopamine receptors (e.g., human D2 (Stormannet al. (1990) Molec. Pharm.37:1-6); rat (Bunzow et al. (1988) Nature336:783-787); and the like); NGF receptors (e.g., human NGF receptors(Johnson et al. (1986) Cell 47:545-554); and the like); serotoninreceptors (e.g., human 5HT1a (Kobilka et al. (1987) Nature 329:75-79);rat 5HT2 (Julius et al. (1990) PNAS 87:928-932); rat 5HTlc (Julius etal. (1988) Science 241:558-564); and the like).

Reporter gene constructs are prepared by operatively linking a reportergene with at least one transcriptional regulatory element. If only onetranscriptional regulatory element is included it must be a regulatablepromoter, At least one of the selected transcriptional regulatoryelements must be indirectly or directly regulated by the activity of theselected cell-surface receptor whereby activity of the receptor can bemonitored via transcription of the reporter genes.

The construct may contain additional transcriptional regulatoryelements, such as a FIRE sequence, or other sequence, that is notnecessarily regulated by the cell surface protein, but is selected forits ability to reduce background level transcription or to amplify thetransduced signal and to thereby increase the sensitivity andreliability of the assay.

Many reporter genes and transcriptional regulatory elements are known tothose of skill in the art and others may be identified or synthesized bymethods known to those of skill in the art.

A reporter gene includes any gene that expresses a detectable geneproduct, which may be RNA or protein. Preferred reporter genes are thosethat are readily detectable. The reporter gene may also be included inthe construct in the form of a fusion gene with a gene that includesdesired transcriptional regulatory sequences or exhibits other desirableproperties.

Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238,Hall et al. (1983) J. Mol. Appl. Gen. 2: 101).

Transcriptional control elements include, but are not limited to,promoters, enhancers, and repressor and activator binding sites,Suitable transcriptional regulatory elements may be derived from thetranscriptional regulatory regions of genes whose expression is rapidlyinduced, generally within minutes, of contact between the cell surfaceprotein and the effector protein that modulates the activity of the cellsurface protein. Examples of such genes include, but are not limited to,the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485),such as c-fos, Immediate early genes are genes that are rapidly inducedupon binding of a ligand to a cell surface protein. The transcriptionalcontrol elements that are preferred for use in the gene constructsinclude transcriptional control elements from immediate early genes,elements derived from other genes that exhibit some or all of thecharacteristics of the immediate early genes, or synthetic elements thatare constructed such that genes in operative linkage therewith exhibitsuch characteristics. The characteristics of preferred genes from whichthe transcriptional control elements are derived include, but are notlimited to, low or undetectable expression in quiescent cells, rapidinduction at the transcriptional level within minutes of extracellularsimulation, induction that is transient and independent of new proteinsynthesis, subsequent shut-off of transcription requires new proteinsynthesis, and mRNAs transcribed from these genes have a shorthalf-life. It is not necessary for all of these properties to bepresent.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 0.1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and instrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” 0 and B books, Corvallis, Oreg., U.S.A., 1977).

Micelles

Recently, the pharmaceutical industry introduced microemulsificationtechnology to improve bioavailability of some lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other things, microemulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom a compound of the present invention and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Particularly preferred amphiphilic carriers are saturated andmonounsaturated polyethyleneglycolyzed fatty acid glycerides, such asthose obtained from fully or partially hydrogenated various vegetableoils. Such oils may advantageously consist of tri- di- and mono-fattyacid glycerides and di- and mono-polyethyleneglycol esters of thecorresponding fatty acids, with a particularly preferred fatty acidcomposition including capric acid 4-10, capric acid 3-9, lauric acid40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%.Another useful class of amphiphilic carriers includes partiallyesterified sorbitan and/or sorbitol, with saturated or mono-unsaturatedfatty acids (SPAN-series) or corresponding ethoxylated analogs(TWEEN-series).

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Polymers

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter alpha., .beta. or gamma.,respectively. Cyclodextrins with fewer than six glucose units are notknown to exist. The glucose units are linked by alpha-1,4-glucosidicbonds. As a consequence of the chair conformation of the sugar units,all secondary hydroxyl groups (at C-2, C-3) are located on one side ofthe ring, while all the primary hydroxyl groups at C-6 are situated onthe other side. As a result, the external faces are hydrophilic, makingthe cyclodextrins water-soluble. In contrast, the cavities of thecyclodextrins are hydrophobic, since they are lined by the hydrogen ofatoms C-3 and C-5, and by ether-like oxygens. These matrices allowcomplexation with a variety of relatively hydrophobic compounds,including, for instance, steroid compounds such as 17.beta.-estradiol(see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113(1994)). The complexation takes place by Van der Waals interactions andby hydrogen bond formation. For a general review of the chemistry ofcyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822(1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C.sub.14 to about C.sub.20).Polymer-derivatized lipids such as PEG-lipids may also be utilized formicelle formation as they will act to inhibit micelle/membrane fusion,and as the addition of a polymer to surfactant molecules decreases theCMC of the surfactant and aids in micelle formation. Preferred aresurfactants with CMCs in the micromolar range; higher CMC surfactantsmay be utilized to prepare micelles entrapped within liposomes of thepresent invention, however, micelle surfactant monomers could affectliposome bilayer stability and would be a factor in designing a liposomeof a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

Release Modifiers

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween.RTM. and Pluronic.RTM. Pore forming agentswhich add microstructure to the matrices (i.e., water soluble compoundssuch as inorganic salts and sugars) are added as particulates. The rangeshould be between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

Combinatorial Libraries

The subject compounds may be synthesized using the methods ofcombinatorial synthesis described in this section. Combinatoriallibraries of the compounds may be used for the screening ofpharmaceutical, agrochemical or other biological or medically-relatedactivity or material-related qualities. A combinatorial library for thepurposes of the present invention is a mixture of chemically relatedcompounds which may be screened together for a desired property; saidlibraries may be in solution or covalently linked to a solid support.The preparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes which need tobe carried out. Screening for the appropriate biological,pharmaceutical, agrochemical or physical property may be done byconventional methods.

Diversity in a library can be created at a variety of different levels.For instance, the substrate aryl groups used in a combinatorial approachcan be diverse in terms of the core aryl moiety, e.g., a variegation interms of the ring structure, and/or can be varied with respect to theother substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93/09668 and WO91/07087; and the Lerner et al. PCT publicationWO93/20242). Accordingly, a variety of libraries on the order of about16 to 1,000,000 or more diversomers can be synthesized and screened fora particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can besynthesized using the subject reactions adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group, e.g.,located at one of the positions of substrate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. In one embodiment, which is particularly suitable for discoveringenzyme inhibitors, the beads can be dispersed on the surface of apermeable membrane, and the diversomers released from the beads by lysisof the bead linker. The diversomer from each bead will diffuse acrossthe membrane to an assay zone, where it will interact with an enzymeassay. Detailed descriptions of a number of combinatorial methodologiesare provided below.

A. Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), e.g.,which can be used to characterize sub-femtomolar amounts of a compound,and to directly determine the chemical constitution of a compoundselected from a combinatorial library. For instance, where the libraryis provided on an insoluble support matrix, discrete populations ofcompounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy is introduced intothe library, the beads are divided into separate groups equal to thenumber of different substituents to be added at a particular position inthe library, the different substituents coupled in separate reactions,and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with sequenceable bio-oligomers

-   -   The principle of using oligonucleotides to encode combinatorial        synthetic libraries was described in 1992 (Brenner et al. (1992)        PNAS 89:5381-5383), and an example of such a library appeared        the following year (Needles et al. (1993) PNAS 90:10700-10704).        A combinatorial library of nominally 7⁷ (=823,543) peptides        composed of all combinations of Arg, Gln, Phe, Lys, Val,        _(D-)Val and Thr (three-letter amino acid code), each of which        was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA        and AC, respectively), was prepared by a series of alternating        rounds of peptide and oligonucleotide synthesis on solid        support. In this work, the amine linking functionality on the        bead was specifically differentiated toward peptide or        oligonucleotide synthesis by simultaneously preincubating the        beads with reagents that generate protected OH groups for        oligonucleotide synthesis and protected NH₂ groups for peptide        synthesis (here, in a ratio of 1:20). When complete, the tags        each consisted of 69-mers, 14 units of which carried the code.        The bead-bound library was incubated with a fluorescently        labeled antibody, and beads containing bound antibody that        fluoresced strongly were harvested by fluorescence-activated        cell sorting (FACS). The DNA tags were amplified by PCR and        sequenced, and the predicted peptides were synthesized.        Following such techniques, compound libraries can be derived for        use in the subject method, where the oligonucleotide sequence of        the tag identifies the sequential combinatorial reactions that a        particular bead underwent, and therefore provides the identity        of the compound on the bead.    -   The use of oligonucleotide tags permits exquisitely sensitive        tag analysis. Even so, the method requires careful choice of        orthogonal sets of protecting groups required for alternating        co-synthesis of the tag and the library member. Furthermore, the        chemical lability of the tag, particularly the phosphate and        sugar anomeric linkages, may limit the choice of reagents and        conditions that can be employed for the synthesis of        non-oligomeric libraries. In preferred embodiments, the        libraries employ linkers permitting selective detachment of the        test compound library member for assay.    -   Peptides have also been employed as tagging molecules for        combinatorial libraries. Two exemplary approaches are described        in the art, both of which employ branched linkers to solid phase        upon which coding and ligand strands are alternately elaborated.        In the first approach (Kerr J M et al. (1993) J Am Chem Soc        115:2529-2531), orthogonality in synthesis is achieved by        employing acid-labile protection for the coding strand and        base-labile protection for the compound strand.    -   In an alternative approach (Nikolaiev et al. (1993) Pept Res        6:161-170), branched linkers are employed so that the coding        unit and the test compound can both be attached to the same        functional group on the resin. In one embodiment, a cleavable        linker can be placed between the branch point and the bead so        that cleavage releases a molecule containing both code and the        compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). In        another embodiment, the cleavable linker can be placed so that        the test compound can be selectively separated from the bead,        leaving the code behind. This last construct is particularly        valuable because it permits screening of the test compound        without potential interference of the coding groups. Examples in        the art of independent cleavage and sequencing of peptide        library members and their corresponding tags has confirmed that        the tags can accurately predict the peptide structure.

2) Non-Sequenceable Tagging: Binary Encoding

-   -   An alternative form of encoding the test compound library        employs a set of non-sequencable electrophoric tagging molecules        that are used as a binary code (Ohlmeyer et al. (1993) PNAS        90:10922-10926). Exemplary tags are haloaromatic alkyl ethers        that are detectable as their trimethylsilyl ethers at less than        femtomolar levels by electron capture gas chromatography (ECGC).        Variations in the length of the alkyl chain, as well as the        nature and position of the aromatic halide substituents, permit        the synthesis of at least 40 such tags, which in principle can        encode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the        original report (Ohlmeyer et al., supra) the tags were bound to        about 1% of the available amine groups of a peptide library via        a photocleavable o-nitrobenzyl linker. This approach is        convenient when preparing combinatorial libraries of        peptide-like or other amine-containing molecules. A more        versatile system has, however, been developed that permits        encoding of essentially any combinatorial library. Here, the        compound would be attached to the solid support via the        photocleavable linker and the tag is attached through a catechol        ether linker via carbene insertion into the bead matrix (Nestler        et al. (1994) J Org Chem 59:4723-4724). This orthogonal        attachment strategy permits the selective detachment of library        members for assay in solution and subsequent decoding by ECGC        after oxidative detachment of the tag sets.    -   Although several amide-linked libraries in the art employ binary        encoding with the electrophoric tags attached to amine groups,        attaching these tags directly to the bead matrix provides far        greater versatility in the structures that can be prepared in        encoded combinatorial libraries. Attached in this way, the tags        and their linker are nearly as unreactive as the bead matrix        itself. Two binary-encoded combinatorial libraries have been        reported where the electrophoric tags are attached directly to        the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031) and        provide guidance for generating the subject compound library.        Both libraries were constructed using an orthogonal attachment        strategy in which the library member was linked to the solid        support by a photolabile linker and the tags were attached        through a linker cleavable only by vigorous oxidation. Because        the library members can be repetitively partially photoeluted        from the solid support, library members can be utilized in        multiple assays. Successive photoelution also permits a very        high throughput iterative screening strategy: first, multiple        beads are placed in 96-well microtiter plates; second, compounds        are partially detached and transferred to assay plates; third, a        metal binding assay identifies the active wells; fourth, the        corresponding beads are rearrayed singly into new microtiter        plates; fifth, single active compounds are identified; and        sixth, the structures are decoded.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 4-Phenyl-piperidine-1,4-dicarboxylic acid mono-tert-butylester

A 1.0 L round-bottom flask was charged with acid (25.0 g; 66.0 mmol),K₂CO₃ (13.7 g; 99.0 mmol), Boc₂O (15.9 g; 73.0 mmol), THF (200 mL) andH₂O (200 mL). The reaction mixture was stirred at 20° C. for 12 h. Thereaction mixture was acidified with 10% HCl to a pH=4 and extracted withEtOAc (500 mL). The organic was dried with MgSO₄ filtered andconcentrated in vacuo to give pure product (20.6 g; 100% yield). ¹H-NMR(300 MHz) δ (ppm) 7.25-7.40 (m; 5H); 3.82 (d; 2H); 2.99 (m; 2H); 2.37(d; 2H); 1.75 (m; 2H); 1.40 (s; 9H).

Example 2 4-Dimethylcarbamoyl-4-phenyl-piperidine-1-carboxylic acidtert-butyl ester

A 250 mL round-bottom flask was charged with acid (6.1 g; 20 mmol),PyBOP (10.4 g; 20 mmol), DMF (100 mL) and NMM (4.4 mL; 40 mmol). Thereaction mixture was stirred at 20° C. for 15 min and then DMAP (240 mg;2.0 mmol) and Me₂NH (12.0 mL; 60.0 mmol) was added. The reaction mixturewas stirred at 20° C. for 12 h. The reaction mixture was diluted with1:1 MTBE/EtOAc (250 mL). The organic was washed with H₂O (250 mL),saturated NaCl (250 mL), dried with MgSO₄, filtered and concentrated invacuo. The crude material was purified by flash chromatography (silicagel hexanes/THF 3:1) to yield pure product (6.0 g; 90% yield). ¹H-NMR(300 MHz) δ (ppm) 7.20-7.40 (m; 5H), 3.98 (d; 2H); 3.23 (t; 2H);2.40-3.00 (bs; 6H); 2.35 (d; 2H); 1.90 (t; 2H); 1.40 (s; 9H). ¹³C-NMR(300 MHz) δ (ppm) 174.1; 155.3; 145.0; 129.3; 128.8; 127.0; 125.2; 79.6;49.8; 41.6; 38.0; 35.6; 28.7.

Example 3 4-Phenyl-piperidine-4-carboxylic acid dimethylamide

A 100 mL round-bottom flask was charged with Boc-protected piperidine(2.0 g; 6.0 mmol) and 50 mL of a 1:1 mixture of TFA and CH₂Cl₂. Thereaction mixture was stirred for 1 h and then concentrated in vacuo. Thecrude material was used without further purification. ¹H-NMR (300 MHz) δ(ppm) 10.61 (s, 3H); 7.90 (bs; 1H); 7.20-7.45 (m, 5H); 3.52 (s; 3H);3.02 (s; 2H); 2.60 (m; 4 H); 2.25 (m; 2H). ¹³C-NMR (300 MHz) δ (ppm)174.6; 141.8; 130.1; 128.4; 124.6; 48.5; 42.8; 39.1; 38.4; 32.2.

Example 4 4-Hydroxymethyl-4-phenyl-piperidine-1-carboxylic acid benzylester

A 500 mL round-bottom flask was charged with acid (25 g; 66.0 mmol),K₂CO₃ (13.7 g; 99.0 mmol), CbzCl (15.9 g; 79.0 mmol), THF (125 mL) andH₂O (125 mL). The reaction mixture was stirred at 20° C. for 12 h. Thereaction mixture was adjusted to pH=4 with 10% HCl and extracted withEtOAc (250 mL). The organic was dried with MgSO₄ filtered andconcentrated in vacuo to give pure product (15.9 g; 71% yield). ¹H-NMR(300 MHz) δ (ppm) 7.25-7.40 (m; 10H); 5.18 (s; 2H); 4.05 (m; 2H); 3.21(m; 2H); 2.55 (m; 2H); 1.95 (m; 2H).

Example 5 4-Hydroxymethyl-4-phenyl-piperidine-1-carboxylic acid benzylester

A 250 mL round-bottom flask was charged with acid (5.0 g; 14.7 mmol) andTHF (75 mL). To this solution was added a 1.0 M solution of BH₃ in THF(44 mL; 44.0 mmol). The reaction mixture was heated to 80° C. for 48 h.The reaction was cooled to room temperature and quenched with 10% HCl.The mixture was adjusted to a pH=6 with 10% NaOH and extracted withEtOAc (100 mL). The organic layer was dried with MgSO₄, filtered andconcentrated in vacuo. The crude material was purified by flashchromatography (silica gel; hexanes/EtOAc 3:1) to give pure product (3.2g; 67% yield). ¹H-NMR (300 MHz) δ (ppm) 7.25-7.40 (m; 10H); 5.16 (s;2H); 3.90 (m; 2H); 3.55 (s; 2H); 3.15 (t; 2H); 2.25 (d; 2H); 1.80 (t;2H).

Example 64-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidine-1-carboxylicacid benzyl ester

A 250 mL round-bottom flask was charged with alcohol (3.2 g; 9.83 mmol),TBDMSCl (2.22 g; 14.8 mmol), imidazole (1.0 g; 14.8 mmol) and DMF (50mL). The reaction mixture was stirred at 20° C. for 12 h. The reactionmixture was diluted with 1:1 MTBE/EtOAc (100 mL). The organic layer waswashed with H₂O (3×100 mL), dried with MgSO₄, filtered and concentratedin vacuo. The crude material was purified by flash chromatography(silica gel; hexanes/EtOAc 3:1) to give pure product (4.63 g; 100%yield). ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 5H); 3.50 (d; 2H); 3.00(m; 2H); 2.80 (m; 2H); 2.20 (m; 2H); 1.95 (m; 2H); 0.82 (s; 9H); −0.13(s; 6H).

Example 7 4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidine

A 100 mL par shaker flask was charged with Cbz-protected amine (4.5 g;10.0 mmol) and MeOH (35 mL). The flask was purged with argon and 10%Pd/C was added (550 mg; 0.5 mmol). The flask was pressurized withhydrogen to 60 psi and agitated for 3 h. The flask was vented and purgedwith argon and the suspension filtered through celite. The celite layerwas washed with MeOH and the combined filtrates concentrated in vacuo togive pure product (3.0 g; 98% yield). ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40(m; 5H); 3.50 (d; 2H); 3.00 (m; 2H); 2.80 (m; 2H); 2.20 (m; 2H); 1.95(m; 2H); 0.82 (s; 9H); −0.13 (s; 6H).

Example 8 General Procedure for the Reductive Amination of4,4-disubstituted piperidines

A 50 mL round-bottom flask was charged with amine (305 mg; 1.0 mmol),THF (5 mL) and aldehyde (2.0 mmol). The reaction mixture was stirred at20° C. for 30 min and the NaBH(OAc)₃ (424 mg; 2.0 mmol) was added. Thereaction mixture was stirred at 20° C. for 12 h. The reaction mixturewas concentrated in vacuo, diluted with DCM (5 mL) and treated withPs-TsNhNH₂ (500 mg) resin by stirring for 30 min. The reaction mixturewas filtered and concentrated in vacuo. The crude material was purifiedby flash chromatography (silica gel; DCM/MeOH 20:1) to give pureproduct.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-(4-chloro-benzyl)-4-phenyl-piperidine

30% yield. ¹H-NMR (300 MHz) δ (ppm) 7.25-7.45 (m; 9 H); 3.65 (s; 2H);3.45 (s; 2H); 3.00 (m; 2H); 2.15-2.45 (m; 4H); 2.05 (s; 2H); 0.80 (s;9H); −0.15 (s; 6H). HRMS (Scan AP+) M/Z=430.0.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-1-thiophen-3-ylmethyl-piperidine

55% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 8H); 3.65 (s; 2H);3.49 (s; 2H); 2.95 (m; 2H); 2.15-2.45 (m; 6H); 0.80 (s; 9H); −0.15 (s;6H). HRMS (Scan AP+) M/Z=401.4.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-(3-methoxy-benzyl)-4-phenyl-piperidine

49% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 8H); 6.90 (m; 1H);3.82 (s; 3H); 3.70 (s; 2H); 3.50 (s; 2H); 3.01 (m; 2H); 2.20-2.60 (m;6H); 0.80 (s; 9H); −0.15 (s; 6H). HRMS (Scan AP+) M/Z=425.8.

Example 9 General Procedure for the Acylation of 4,4-disubstitutedpiperidines

To a 100 mL round-bottom flask was added acid (10.2 mmol), PyBOP (5.3 g;10.2 mmol), DMF (50 mL) and NMM (2.24 mL; 20.4 mmol). The reactionmixture was stirred for 15 min and then DMAP (245 mg; 2.0 mmol) andamine (3.11 g; 10.2 mmol) was added. The reaction mixture was stirred at20° C. for 12 h. The reaction mixture was diluted with 1:1 MTBE/EtOAc(50 mL). The organic layer was washed with H₂O (100 mL), dried withMgSO₄, filtered and concentrated in vacuo. The crude material waspurified by flash chromatography (silica gel; hexanes/EtOAc 3:1) to givepure product.

[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-[1-(4-chloro-phenyl)-cyclobutyl]-methanone

69% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 9H); 4.38 (m; 1H);3.35 (s; 2H); 3.19 (m; 1H); 2.65-3.00 (m; 4H); 2.40 (m; 2H); 2.15 (m;1H); 1.95 (m; 1H); 1.82 (m; 4H); 0.82 (s; 9H); −0.18 (s; 6H). HRMS (ScanAP+) M/Z=497.9.

[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-[1-(4-methoxy-phenyl)-cyclopentyl]-methanone

50% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 5H); 7.17 (d; 2H);6.85 (d; 2H); 4.40 (m; 1H); 3.80 (s; 3H); 3.41 (m; 1H); 3.33 (s; 2H);2.85 (m; 1H); 2.75 (m; 1H); 2.48 (m; 1H); 2.35 (m; 1H); 1.98 (m; 3H);1.78 (m; 5H); 1.38 (m; 2H); 0.81 (s; 9H); −0.19 (s; 6H). HRMS (Scan AP+)M/Z=507.9.

[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-[1-(3-fluoro-phenyl)-cyclopentyl]-methanone

54% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 5H); 7.17 (m; 1H);6.98 (m; 3H); 4.40 (m; 1H); 3.40 (m; 1H); 3.33 (s; 2H); 2.85 (m; 1H);2.75 (m; 1H); 2.51 (m; 1H); 2.37 (m; 1H); 2.18 (m; 1H); 1.98 (m; 3H);1.75 (m; 5H); 1.40 (m; 1H); 0.81 (s; 9H); −0.19 (s; 6H). HRMS (Scan AP+)M/Z=496.0.

1-[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-2-(4-chloro-phenyl)-2-methyl-propan-1-one

45% yield. ¹H-NMR (300 MHz) δ (ppm) 7.10-7.45 (m; 9H); 4.42 (m; 1H);3.38 (s; 2H); 3.26 (m; 1H); 2.83 (m; 1H); 2.75 (m; 1H); 1.91 (s; 3H);1.62 (s; 3H); 1.50 (m; 4H); 0.81 (s; 9H); −0.19 (s; 6H). HRMS (Scan AP+)M/Z=486.9.

Example 10 General Procedure for the Reduction of 4,4-disubstitutedpiperidine amides

To a 50 mL round-bottom flask was added a 1.0 M solution of LAH in THF(21.0 mL; 21.0 mmol) and THF (10 mL). The solution was cooled to 0° C.and a solution of amide (7.0 mmol) in THF (5 mL) was added. The reactionmixture was heated to reflux and stirred for 4 h. The reaction mixturewas cooled to 0° C. and quenched with 10% HCl. The mixture was adjustedto pH=9 with 10% NaOH and extracted with EtOAc. The organic layer wasdried with MgSO₄, filtered and concentrated in vacuo. The crude materialwas purified by flash chromatography (silica gel; hexanes/EtOAc 4:1) togive pure product.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-[1-(4-chloro-phenyl)-cyclobutylmethyl]-4-phenyl-piperidine (16)

86% yield. ¹H-NMR (300 MHz) δ (ppm) 7.20-7.35 (m; 7H); 7.10 (d; 2H);3.42 (s; 2H); 2.58 (s; 2H); 2.10-2.35 (m; 4H); 2.0 (m; 2H); 1.83 (m;2H); 0.81 (s; 9H); −0.18 (s; 6H). HRMS (Scan AP+) M/Z=484.2.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-[1-(4-methoxy-phenyl)-cyclopentylmethyl]-4-phenyl-piperidine (17)

100% yield. ¹H-NMR (300 MHz) δ (ppm) 7.15-7.35 (m; 7H); 6.90 (d; 2H);3.82 (s; 3H); 3.42 (s; 2H); 2.15 (m; 2H); 2.12 (s; 2H); 1.75-2.05 (m;14H); 0.81 (s; 9H); −0.18 (s; 6H). HRMS (Scan AP+) M/Z=493.9.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-[1-(3-fluoro-phenyl)-cyclopentylmethyl]-4-phenyl-piperidine (18)

66% yield. ¹H-NMR (300 MHz) δ (ppm) 7.15-7.35 (m; 6H); 7.05 (m; 2H);6.94 (m; 1H); 3.42 (s; 2H); 2.35 (s; 2H); 1.70-2.20 (m; 16H); 0.81 (s;9H); −0.18 (s; 6H). HRMS (Scan AP+) M/Z=481.9.

4-(tert-Butyl-dimethyl-silanyloxymethyl)-1-[2-(4-chloro-phenyl)-2-methyl-propyl]-4-phenyl-piperidine (19)

60% yield. ¹H-NMR (300 MHz) δ (ppm) 7.15-7.35 (m; 9H); 3.42 (s; 2H);2.35 (m; 4H); 2.20 (m; 2H); 1.98 (m; 2H); 1.85 (m; 2H); 1.29 (s; 6H);0.81 (s; 9H); −0.18 (s; 6H). HRMS (Scan AP+) M/Z=471.9.

Example 112-[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-1-(4-chloro-phenyl)-ethanone

A 100 mL round-bottom flask was charged with amine (715 mg; 2.54 mmol),cesium carbonate (2.49 g; 7.63 mmol), acetonitrile (20 mL) anda-bromoketone (891 mg; 3.82 mmol). The reaction mixture was heated to60° C. and stirred for 1 h. The reaction mixture was cooled to roomtemperature and diluted with EtOAc (50 mL). The organic layer was washedwith saturated NH₄Cl (50 mL), saturated NaCl (50 mL), dried with MgSO₄,filtered and concentrated in vacuo. The crude material was purified byflash chromatography (silica gel; hexanes/EtOAc 3:1 to 1:1) to give pureproduct (715 mg; 61% yield). ¹H-NMR (300 MHz) δ (ppm) 7.99 (d; 2H); 7.42(d; 2H); 7.36 (d; 4H); 7.24 (m; 1H); 3.66 (s; 2H); 3.49 (s; 2H); 2.80(m; 2H); 2.38 (m; 2H); 2.22 (M; 2H); 2.05 (m; 2H); 0.82 (s; 9H); −0.13(s; 6H). ¹³C-NMR (300 MHz) δ (ppm) 196.2; 143.9; 139.8; 134.7; 130.0;129.0; 128.3; 127.8; 126.1; 72.6; 65.5; 50.6; 42.2; 31.6; 26.1; 18.5;−5.5.

Example 122-[4-(tert-Butyl-dimethyl-silanyloxymethyl)-4-phenyl-piperidin-1-yl]-1-(4-chloro-phenyl)-ethanol

A 100 mL round-bottom flask was charged with ketone (715 mg; 1.56 mmol),methanol (20 mL) and sodium borohydride (65 mg; 1.71 mmol). The reactionmixture was and stirred at 20 C. for 1 h. The reaction mixture wascooled to room temperature and diluted with EtOAc (100 mL). The organiclayer was washed with 5% HCl (100 mL), saturated NaCl (100 mL), driedwith MgSO₄, filtered and concentrated in vacuo. The crude material waspurified by flash chromatography (silica gel; DCM with 2% 2.0 M NH₃ inEtOH) to give pure product (559 mg; 78% yield). ¹H-NMR (300 MHz) δ (ppm)7.38 (d; 2H); 7.36 (d; 2H); 7.31 (s; 4H); 7.24 (m; 1H); 4.72 (dd; 1H);3.50 (s; 2H); 2.99 (dd; 1H); 2.62 (m; 1H); 2.58 (m; 1H); 2.40 (m; 2H);2.22 (m; 3H); 2.05 (m; 2H); 0.82 (s; 9H); −0.13 (s; 6H). ¹³C-NMR (300MHz) δ (ppm) 143.8; 141.2; 133.2; 128.7; 128.4; 127.8; 127.4; 126.2;72.5; 68.3; 66.4; 51.2; 49.1; 42.7; 31.9; 31.7; 26.1; 18.5; −5.5.

Example 13 General Procedure for the Deprotection of a Silyl EtherComprised by a 4,4-disubstituted piperidine

To a 50 mL round-bottom flask was added TBDMS ether (6.0 mmol), THF (20mL) and a solution of 1.0 M TBAF in THF (12.0 mL, 12.0 mmol). Thereaction mixture was stirred at 50° C. for 12 h. The reaction mixturewas concentrated in vacuo and the crude material was purified by flashchromatography (silica gel; EtOAc) to give pure product.

[1-(4-Chloro-benzyl)-4-phenyl-piperidin-4-yl]-methanol (1):

¹H-NMR (300 MHz) δ (ppm) 7.25-7.45 (m; 9 H); 3.63 (s; 2H); 3.42 (s; 2H);2.70 (m; 2H); 2.25 (m; 4H); 1.98 (t; 2H). HRMS (Scan AP+) M/Z=316.5.

(4-Phenyl-1-thiophen-3-ylmethyl-piperidin-4-yl)-methanol (2):

¹H-NMR (300 MHz) δ (ppm) 7.05-7.45 (m; 8H); 3.65 (s; 2H); 3.52 (s; 2H);2.75 (m; 2H); 2.30 (m; 4H); 2.00 (t; 2H). HRMS (Scan AP+) M/Z=287.9.

[1-(3-Methoxy-benzyl)-4-phenyl-piperidin-4-yl]-methanol (3)

¹H-NMR (300 MHz) δ (ppm) 7.20-7.45 (m; 5H); 6.80-6.95 (m; 4H); 3.81 (s;3H); 3.60 (s; 2H); 3.50 (s; 2H); 2.75 (m; 2H); 2.30 (m; 4H); 2.00 (t;2H). HRMS (Scan AP+) M/Z=312.1.

{1-[1-(4-Chloro-phenyl)-cyclobutylmethyl]-4-phenyl-piperidin-4-yl}-methanol(4):

¹H-NMR (300 MHz) δ (ppm) 7.20-7.45 (m; 7H); 7.15 (d; 2H); 3.55 (s; 2H);3.60 (s; 4H); 1.95-2.40 (m; 12H); 1.60-1.90 (m; 4H). ¹³C-NMR (300 MHz) δ(ppm) 143.4; 139.8; 132.9; 129.5; 129.4; 127.9; 127.3; 127.1; 72.0;67.3; 66.8; 50.7; 44.3; 41.9; 33.7; 28.6; 16.3. HRMS (Scan AP+)M/Z=369.9.

{1-[1-(4-Methoxy-phenyl)-cyclopentylmethyl]-4-phenyl-piperidin-4-yl}-methanol(6):

¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 6H); 6.90 (d; 2H); 3.82 (s; 3H);3.55 (s; 2H); 2.35 (s; 2H); 2.20 (m; 2H); 2.10 (m; 2H); 2.00 (m; 4H);1.50-1.80 (m; 8H). HRMS (Scan AP+) M/Z=380.2.

{1-[2-(4-Chloro-phenyl)-2-methyl-propyl]-4-phenyl-piperidin-4-yl}-methanol(11):

¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 9H); 3.58 (s; 2H); 2.35 (m; 4H);2.20 (m; 2H); 2.05 (m; 2H); 1.80 (m; 2H); 1.28 (s; 6H). ¹³C-NMR (300MHz) δ (ppm) 143.9; 140.1; 133.3; 129.5; 129.4; 127.9; 127.4; 127.0;71.9; 69.1; 67.3; 52.4; 41.9; 40.3; 37.8; 28.7; 38.1. HRMS (Scan AP+)M/Z=357.9.

1-(4-Chloro-phenyl)-2-(4-hydroxymethyl-4-phenyl-piperidin-1-yl)-ethanol(12):

¹H-NMR (300 MHz) δ (ppm) 7.20-7.45 (m; 9H); 4.73 (dd, 1H); 3.61 (s; 2H);2.98 (m; 1H); 2.61 (m; 2H); 2.20-2.50 (m; 5H); 2.0 (m; 2H). HRMS (ScanAP+) M/Z=345.5.

Example 14 4-Phenethylcarbamoyl-4-phenyl-piperidine-1-carboxylic acidbenzyl ester

To a 250 mL round-bottom flask was added acid (5.0 g; 14.7 mmol), PyBOP(7.65 g; 14.7 mmol), DMF (75 mL) and NMM (3.2 mL; 29.5 mmol). Thereaction mixture was stirred for 15 min and then DMAP (180 mg; 1.5 mmol)and amine (1.9 mL; 14.7 mmol) was added. The reaction mixture wasstirred at 20° C. for 12 h. The reaction mixture was diluted with 1:1MTBE/EtOAc (200 mL). The organic layer was washed with H₂O (2×200 mL),dried with MgSO₄, filtered and concentrated in vacuo. The crude materialwas purified by flash chromatography (silica gel; hexanes/EtOAc 1:1) togive pure product (6.2 g; 95% yield). HRMS (Scan AP+) M/Z=442.9.

Example 15 4-(Phenethylamino-methyl)-4-phenyl-piperidine-1-carboxylicacid benzyl ester

A 250 mL round-bottom flask was charged with amide (6.2 g; 14.0 mmol)and THF (100 mL). To this solution was added a 1.0 M solution of BH₃ inTHF (42 mL; 42.0 mmol). The reaction mixture was heated to 80° C. for 18h. The reaction was cooled to room temperature and quenched with 10%HCl. The mixture was adjusted to a pH=9 with 10% NaOH and extracted withEtOAc (100 mL). The organic layer was dried with MgSO₄, filtered andconcentrated in vacuo. The crude material was used without purification.HRMS (Scan AP+) M/Z=428.2.

Example 164-[(tert-Butoxycarbonyl-phenethyl-amino)-methyl]-4-phenyl-piperidine-1-carboxylicacid benzyl ester

A 250 mL round-bottom flask was charged with amine (14.0 mmol), K₂CO₃(2.9 g; 21.0 mmol), Boc₂O (3.41 g g; 15.4 mmol), THF (50 mL) and H₂O (50mL). The reaction mixture was stirred at 20° C. for 12 h. The reactionmixture was acidified with 10% HCl to a pH=4 and extracted with EtOAc(500 mL). The organic was dried with MgSO₄ filtered and concentrated invacuo to give pure product (3.65 g; 49% yield). ¹H-NMR (300 MHz) δ (ppm)7.00-7.40 (m; 15H); 5.11 (s; 2H); 3.97 (m; 2H); 3.15 (m; 2H); 2.80 (m;2H): 2.60 (m; 4H); 2.20 (m; 2H); 1.92 (m:2H); 1.45 (s; 9H). HRMS (ScanAP+) M/Z=429.1.

Example 17 Phenethyl-(4-phenyl-piperidin-4-ylmethyl)-carbamic acidtert-butyl ester

A 100 mL par shaker flask was charged with Cbz-protected amine (3.65 g;6.9 mmol) and MeOH (35 mL). The flask was purged with argon and 10% Pd/Cwas added (734 mg; 0.69 mmol). The flask was pressurized with hydrogento 60 psi and agitated for 12 h. The flask was vented and purged withargon and the suspension filtered through celite. The celite layer waswashed with MeOH and the combined filtrates concentrated in vacuo togive pure product (2.88g; 100% yield). ¹H-NMR (300 MHz) δ (ppm)7.20-7.40 (m; 9H); 7.00 (m; 1H); 3.28 (s; 1H); 3.18 (s; 1H); 2.98 (t;2H): 2.50-2.90 (m; 6H); 2.20 (m; 2H); 1.92 (m; 2H); 1.45 (s; 9H). HRMS(Scan AP+) M/Z=395.0.

Example 18{1-[1-(4-Methoxy-phenyl)-cyclopropanecarbonyl]-4-phenyl-piperidin-4-ylmethyl}-phenethyl-carbamic acid tert-butyl ester

To a 250 mL round-bottom flask was added acid (1.0 mmol), PyBOP (520 mg;1.0 mmol), DMF (7 mL) and NMM (220 μL; 2.0 mmol). The reaction mixturewas stirred for 15 min and then DMAP (12 mg; 0.1 mmol) and amine (396mg; 1.0 mmol) was added. The reaction mixture was stirred at 20° C. for12 h. The reaction mixture was diluted with 1:1 MTBE/EtOAc (50 mL). Theorganic layer was washed with H₂O (2×50 mL), dried with MgSO₄, filteredand concentrated in vacuo. The crude material was purified by flashchromatography (silica gel; hexanes/EtOAc 2:1) to give pure product (51%yield). ¹H-NMR (300 MHz) δ (ppm) 7.38 (t; 2H); 7.25 (m; 6H); 7.11 (d;2H); 6.98 (m; 2H); 6.81 (d; 2H); 4.30 (m; 2H); 3.90 (m; 2H); 3.80 (s;2H); 3.23 (s; 2H); 3.05 (m; 2H); 2.65 (m; 2H); 2.50 (m; 2H); 2.20 (m;2H); 2.02 (m; 2H); 1.41 (s; 9H). HRMS (Scan AP+) M/Z=567.8.

Example 19{1-[1-(4-Methoxy-phenyl)-cyclopropylmethyl]-4-phenyl-piperidin-4-ylmethyl}-phenethyl-carbamic acid S-butyl ester

To a 25 mL round-bottom flask was added amide (0.31 mmol), and toluene(5 mL) and REDAL (325 μL; 1.08 mmol). The reaction mixture was stirredat 20 C. for 12 h. The reaction mixture was cooled to 0° C. and quenchedwith 10% NaOH (1 mL) and extracted with EtOAc. The organic layer wasdried with MgSO₄, filtered and concentrated in vacuo. The crude materialwas purified by flash chromatography (silica gel; hexanes/EtOAc 2:1) togive pure product (118 mg; 85% yield). HRMS (Scan AP+) M/Z=554.7.

Example 20{1-[1-(4-Methoxy-phenyl)-cyclopropylmethyl]-4-phenyl-piperidin-4-ylmethyl}-phenethyl-amine(5)

To a 25 mL round-bottom flask was added N-Boc amine (1.0 mmol), DCM (2.5mL) and TFA (0.5 mL). The reaction mixture was stirred at 20° C. for 1h. The reaction mixture was concentrated in vacuo to give pure product(100% yield). ¹H-NMR (300 MHz) δ (ppm) 7.00-7.80 (m; 14H); 3.40 (s; 2H);3.35 (m; 2H); 3.19 (m; 4H); 2.90 (t; 2H); 2.55 (m; 4H); 2.30 (m; 4H);2.00 (m; 2H). HRMS (Scan AP+) M/Z=567.8.

Example 211-[1-(4-Chloro-phenyl)-cyclobutanecarbonyl]-4-phenyl-piperidine-4-carboxylicacid dimethylamide

To a 250 mL round-bottom flask was added acid (1.41 g; 6.6 mmol), PyBOP93.48 g; 6.6 mmol), DMF (100 mL), and NMM (1.33 mL; 12.1 mmol). Stir forat 20 C. for 10 min. To the reaction mixture was added amine (1.41 g;6.0 mmol. The reaction mixture was stirred at 20° C. for 12 h. Thereaction mixture was diluted with 1:1 MTBE/EtOAc (250 mL). The organiclayer was washed with H₂O (200 mL), dried with MgSO₄, filtered andconcentrated in vacuo. The crude material was purified by flashchromatography (silica gel; hexanes/EtOAc 3:1) to give pure product (894mg; 35% yield). ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 7H); 7.11 (d;2H); 4.60 (dt; 1H); 3.21 d; 2H); 2.95 (m; 6H); 2.50 (m; 4H); 2.30 (m;2H); 2.00 (m; 4H); 1.10 (m; 1H). HRMS (Scan AP+) M/Z=425.3.

Example 22{1-[1-(4-Chloro-phenyl)-cyclobutylmethyl]-4-phenyl-piperidin-4-ylmethyl}-dimethyl-amine(9)

To a 50 mL round-bottom flask was added bis-amide (894 mg; 2.1 mmol),THF (25 mL). The reaction mixture was cooled to 0° C. and DIBAL-H (12.6mL; 12.6 mmol) was added. The reaction mixture was warmed to 20° C. andstirred for 48 h. The reaction mixture was diluted with EtOAc (70 mL)and quenched with H₂O (10 mL) and solid Na₂SO₄ (3 g) and stirred for 15min. The reaction mixture was filtered through silica gel andconcentrated in vacuo. The crude material was purified by preparatoryHPLC (C-18, MeCN/H₂O 75:25) to give pure product (724 mg; 89% yield).¹H-NMR (300 MHz) δ (ppm) 7.40-7.60 (m; 5H); 7.24 (d; 2H); 7.18 (d; 2H);3.45 (s; 2H); 3.30 (s; 2H); 3.21 (d; 2H); 2.92 (d; 2H); 2.60 (m; 2H);2.50 (m; 4H); 2.47 (bs; 6H); 2.30 (m; 2H); 2.05 (m; 1H); 1.97 (m; 1H).HRMS (Scan AP+) M/Z=396.8.

Example 231-[1-(4-Chloro-phenyl)-cyclobutylmethyl]-4-methoxymethyl-4-phenyl-piperidine(10)

To a 25 mL round-bottom flask was added NaH (68 mg; 2.70 mmol), THF (10mL), alcohol (200 mg; 0.54 mmol) and methyl iodide (337 μL, 5.40 mmol).The reaction mixture was stirred at reflux for 12 h. The reactionmixture was quenched with saturated NH₄Cl (50 mL) and extracted withEtOAc (50 mL). The organic layer was washed with water (50 mL),saturated NaCI (50 mL), dried with MgSO₄, filtered and concentrated invacuo. The crude material was purified by flash chromatography (silicagel; hexanes/EtOAc 6:1 to 3:1) to give pure product (94 mg; 45% yield).¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m; 7H); 7.16 (d; 2H); 3.33 (s; 2H);3.22 (s; 3H); 2.60 (s; 2H); 2.30 (m; 5H); 2.24 (m; 5H); 2.03 (m; 2H);1.86 (m; 2H). ). ¹³C-NMR (300 MHz) δ (ppm) 148.7; 145.0; 131.1; 128.4;128.0; 127.8; 127.4; 126.1; 81.9; 68.7; 59.6; 52.0; 47.3; 40.8; 32.7;31.9; 16.3. HRMS (Scan AP+) M/Z=384.5.

Example 24 Acetic Acid1-[2-acetoxy-2-(4-chloro-phenyl)-ethyl]-4-phenyl-piperidin-4-ylmethylester (13)

To a 25 mL round-bottom flask was added diol (300 mg; 0.87 mmol), DCM (5mL), DMAP (10.6 mg; 0.09 mmol), iPr₂NEt (755 μL; 4.3 mmol) and Ac₂O (205μL; 2.17 mmol). The reaction mixture was stirred at 20° C. for 3 h. Thereaction mixture was diluted with EtOAc (50 mL) and washed withsaturated NaHCO₃ (50 mL), saturated NaCl (50 mL), dried with MgSO₄,filtered and concentrated in vacuo. The crude material was purified byflash chromatography (silica gel, DCM with 2% 2.0 M NH₃ in EtOH) to givepure product (263 mg; 70% yield). ¹H-NMR (300 MHz) δ (ppm) 7.20-7.40 (m;9H); 5.90 (dd; 1H); 4.08 (s; 2H); 3.79 (dd; 1H); 3.70 (m; 2H); 2.49 (dd;1H); 2.40 (m; 2H); 2.20 (m; 2H); 2.10 (s; 3H); 1.98 (s; 3H); 1.90 (m;2H). HRMS (Scan AP+) M/Z=396.8.

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound represented by A:

wherein R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl,heteroaryl, acyl, or sulfonyl; R₁ represents aryl, or heteroaryl; R₂represents RO-alkyl, (R)₂N-alkyl, RS-alkyl, RO-cycloalkyl,(R)₂N-cycloalkyl, or RS-cycloalkyl; R₃ represents H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F; R₄ represents H,alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;R₅ represents an aryl or heteroaryl group; R₃ and R₄ may be connectedthrough a covalent bond; n is 0, 1, or 2; and the stereochemicalconfiguration at any stereocenter of a compound represented by A is R,S, or a mixture of these configurations.
 2. The compound of claim 1,wherein R₅ represents phenyl.
 3. The compound of claim 1, wherein R₁represents aryl.
 4. The compound of claim 1, wherein R₂ representsRO-alkyl.
 5. The compound of claim 1, wherein n is 1; and R₃ representsH, alkyl, or F.
 6. The compound of claim 1, wherein n is 1; and R₄represents H, alkyl, or F.
 7. The compound of claim 1, wherein R₅represents phenyl; and R₁ represents aryl.
 8. The compound of claim 1,wherein R₅ represents phenyl; R₁ represents aryl; and R₂ representsRO-alkyl.
 9. The compound of claim 1, wherein n is 1; R₅ representsphenyl; R₁ represents aryl; R₂ represents RO-alkyl; and R₃ represents H,alkyl, or F.
 10. The compound of claim 1, wherein n is 1; R₅ representsphenyl; R₁ represents aryl; R₂ represents RO-alkyl; R₃ represents H,alkyl, or F; and R₄ represents H, alkyl, or F.
 11. The compound of claim1, wherein n is 0; and R₅ represents phenyl.
 12. The compound of claim1, wherein n is 0; and R₅ represents thiophene. 13-44. (canceled)
 45. Aformulation, comprising a compound of claim 1; and a pharmaceuticallyacceptable excipient.
 46. The formulation of claim 45, wherein saidpharmaceutically acceptable excipient is selected from the groupconsisting of cyclodextrins, celluloses, liposomes, micelle formingagents, and polymeric carriers.
 47. A method of modulating the activityof a dopamine, serotonin, or norepinephrine receptor or transporter in amammal, comprising the step of administering to said mammal atherapeutically effective amount of a compound represented by A:

wherein R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl,heteroaryl, acyl, or sulfonyl; R₁ represents aryl, heteroaryl, aralkyl,or heteroaralkyl; R₂ represents alkyl, RO-alkyl, (R)₂N-alkyl, RS-alkyl,cycloalkyl, RO-cycloalkyl, (R)₂N-cycloalkyl, RS-cycloalkyl, alkenyl,aryl, or heteroaryl; R₄ represents independently for each occurrence H,alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;R₅ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F; any geminal orvicinal pairs of R₄ and R₅ may be connected through a covalent bond; nis independently for each occurrence 0, 1, 2, 3, or 4; and thestereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations. 48-76.(canceled)
 77. A method of treating a mammal suffering from addiction,anxiety, depression, sexual dysfunction, hypertension, migraine,Alzheimer's disease, obesity, emesis, psychosis, analgesia,schizophrenia, Parkinson's disease, restless leg syndrome, sleepingdisorders, attention deficit hyperactivity disorder, irritable bowelsyndrome, premature ejaculation, menstrual dysphoria syndrome, urinaryincontinence, inflammatory pain, neuropathic pain, Lesche-Nyhanedisease, Wilson's disease, or Tourette's syndrome, comprising the stepof administering to said mammal a therapeutically effective amount of acompound represented by A:

wherein R represents H, alkyl, aralkyl, cycloalkyl, alkenyl, aryl,heteroaryl, acyl, or sulfonyl; R₁ represents aryl, heteroaryl, aralkyl,or heteroaralkyl; R₂ represents alkyl, RO-alkyl, (R)₂N-alkyl, RS-alkyl,cycloalkyl, RO-cycloalkyl, (R)₂N-cycloalkyl, RS-cycloalkyl, alkenyl,aryl, or heteroaryl; R₄ represents independently for each occurrence H,alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F;R₅ represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —OR, or F; any geminal orvicinal pairs of R₄ and R₅ may be connected through a covalent bond; nis independently for each occurrence 0, 1, 2, 3, or 4; and thestereochemical configuration at any stereocenter of a compoundrepresented by A is R, S, or a mixture of these configurations. 78-91.(canceled)
 92. The compound of claim 1, wherein R₁ representsheteroaryl.